Amino acid synthesis



United States Patent 2,921,059 AMINO ACIDSYNTHESIS Robert C. Good, Northbrook, and Irwin C. Gunsalus, Urbana, Ill., assignors to International Minerals & Chemical Corporation, a corporation of New York No Drawing. Application June 27, 1956 Serial No. 594,091

16 Claims. (Cl. 195-29) This invention is concerned with the synthesis of alpha-amino carboxylic acids, and more particularly with the preparation of such acids by enzymatic treatment of the corresponding alpha-keto carboxylic acids,

Glutamic acid is a well-known constituent of proteins, and is widely used as a flavor-intensifying agent in the form of the monosodium salt of its L-enantiomorph. Substantially all of the L-glutamic acid utilized for the preparation of monosodium L-glutamate is obtained at the present time from natural sources such as Wheat gluten and sugar beet residues, for example, from Stefiens filtrate. While it is known that L-glutamic acid may be synthetically produced, most processes for synthesizing glutamic acid produce the racemic mixture, DL-glutamic acid, which must be further processed (resolved) to produce L-glutamic acid. No process has yet been commercially proved for, effecting such resolution.

It is known that glutamic dehydrogenase from liver,

kidney, and heart tissues reversibly oxidizes L-glutamic acid to alpha-ketoglutaric acid and ammonia in the presence of coenzyme I or eoenzyme II, the initial reaction probably being the formation of iminoglutaric acid, which then hydrolyzes spontaneously to alpha-ketoglutaric acid and ammonia (Sumner and Somers, Chemistry and Methods of Enzymes, New York: Academic Press, Inc., Third Edition (1953), page 269). Thus, it is possible to prepare L-glutamic acid from alpha-ketoglutaric acid and ammonia by the action of such glutamic dehydrogenase (Hunter and Hixon, J. Biol. Chem., 181 (1949), page 67; cited by Sumner and Myrback, The Enzymes, New York: Academic Press, Inc. (1953), vol. 11, part 1, page 308). Clutamicdehydrogenase from yeast exhibits a similar action (Sumner and Somers, op. cit., page 268).

The present invention is an improvement in the enzymatic production of L-alpha-amino carboxylic acids from the corresponding alpha-keto carboxylic acids.

It is an object of the invention to provide an improved process for preparing L-alpha-amino carboxylates from the corresponding alpha-keto carboxylates.

It is another object of the invention to prepare L- alpha-amino carboxylic acids by the yeast fermentation of the corresponding alpha-keto carboxylic acids in an improved manner.

Another object of the invention is to provide an improved process for converting alpha-ketoglutaric acid to L-glutamic acid.

. Another object of the invention is to improve the yield of L-glutamic acid obtained in the fermentation of alphaketo-glutarie acid with yeast.

Still another object of this invention is to provide a commercially practicable procedure for preparing L- glutamic acid by enzymatic conversion of alpha-ketoglutaric acid.

We have now discovered that the enzymatic conversion of alphaketo carboxylic acids to the corresponding L-alpha-amino carboxylic acids is advantageously'carried out with whole yeast cells in the presence of a lipid solvent, the reaction medium being otherwise of con ventional composition. The lipids are extracted from the yeast cells by the solvent, and the cells are greatly improved in permeability in this way. The interchange of enzymes and reactants between the yeast cells and the reaction medium is, in our belief, greatly facilitated thereby, although it is understood that this is only a reasonable theory of the reaction mechanism and we do not wish to be bound thereby. In any event, the yield of the desired product is substantially greater in our process than in the processes of the prior art.

We have further discovered that the yield of the desired product is considerably improved if the fermentation medium, prior to the introduction of alpha-keto carboxylic acid, is subjected to a preincubation period of about 1 to 10 hours, preferably around 2 hours, under the fermentation conditions thereafter to be employed. It is our belief that the observed improvement results from a build-up of diphosphopyridine nucleotide (DPN) during the preincubation period, although in this respect also we do not wish to be restricted in any way by the suggested mechanism.

We have additionally discovered that the fermentation is advantageously carried out in the absence of free oxygen, and preferablyunder an atmosphere of carbon dioxide.

The term alpha-amino carboxylate as used herein is intended to refer to alpha-amino carboxylic acid, alphaamino dicarboxylic acid, and the alkali metal and alkaline earth metal salts of these materials. Similarly, the term alpha-keto carboxylate asused herein is intended to refer to alpha-keto carboxylic 'acid, alpha-keto dicarboxylicacid, and the alkali metal and alkaline earth metal salts of these acids. a

The term fermentation as used in this description and attached claims is intended to include any process in which organisms are employed to catalyze the conversion of a substrate into one or more desired products.

While alpha-ketoglutarate' is the preferred alpha-keto carboxylate for our process, yielding L-glutamic acid, other alpha-keto carboxylates may be employed to ad vantage for the synthesis of other alpha-amino carboxylf ates; for example, oxalacetate to produce aspartic acid, pyruvate to produce alanine, methyl-alpha-ketobutyric acid to produce valine, etc. .Also alpha-ketoparboxylate precursor compounds such as citrate, isocitrate, or aconitate for alpha-ketoglutarate; fumarate or malate for oxalacetate; or the fermentable carbohydrates as precursors of alpha-ketoglutarate. or oxalacetate may be employed to replace the alpha-keto carboxylate wholly or partially in conducting our novel process. The use of furnarate and malate are not limited only to the production of oxalacetate, but in some instances these substances will be converted into aspartic acid without first producing oxalacetate.

In accordance with one embodiment of this invention, an alpha-keto carboxylate is treated with whole yeast cells in a suitable aqueous medium containing a lipid solvent until the alpha-keto earboxylate is substantially converted to the corresponding alpha-amino carboxylate. Any lipid solvent may be utilized, but carbon tetrachloride is particularly preferred. Other suitable lipid solvents include ethylene dichloride, trichloroethylene,

The foregoing example illustrates successful fermentations in which the fermentation medium included both whole yeast cells and lipid solvent. The following test illustrates the comparatively poor results obtained when the fermentation is carried out in the presence of a lipid solvent (carbon tetrachloride) extract of yeast cells, but in the absence of the yeast cells themselves,

An aqueous fermentation medium having the composition described in Example I and containing carbon tetrachloride as the lipid solvent was incubated for 2 hours at 37 C., then centrifuged to remove the yeast cells.

The supernatant liquor was collected,- commingled with r 60 ml. of aqueous 2% alpha-kctoglutaric acid solution (adjusted to pH 7.6 with NaOH), and returned to the 37 C. incubator. Samples were withdrawn from time to time for analysis, with the following results:

L-Glutarnic Acid a-KEtO- Lipid Solvent Time, glutarate hr. adde Found, Syn- ConvernMJml. nMJml. thesized, sion,

uMJml. percent r 2 5 12'? 5e "11'? 91 5 82 1912 12: 4 15: t 24 19. 2 12. 4 15.].

Example 111 The following test demonstrates the advantage of preincubating the fermentation medium before adding alphaketoglutarate. Two portions of a medium were prepared as described in Example I and adjusted to pH 7.6 with NaOH. One portion was preincubated for 2 hours at 37 C., while the second portion was not preincubated. To each portion were added 82 ,uM./ml. of alpha-ketoglutarate, and the primary incubation was carried out at 37 C. Samples were removed for analysis at 0, 5, 10, and 24 hours. The results were as follows:

L-Glutamic Acid a-Keto- Treatment Time, glutarate hr. added, Found, Syn- ConveruMJml. MJml. thesized, sion,

" lVL/ml percent 0 22-; 5 5. 3 Pre'mcubated 10 82 v 63.8 40.7 49.6 24 73.4 50.3 61.3

2 e No Preincubation 10 82 24 62.6 40.9 49.9

Example IV The following test demonstrates the advantage of carrying out the fermentation under an atmosphere of carbon dioxide. An aqueous fermentation medium having the following composition was prepared in duplicate.

0.2 M dibasic ammonium phosphate (pH 7.6 with HCl) ml 1 15.0 MnSO, (0.1 M) l 0.5 Sucrose g 1.0 Water ml 4.5 Carbon tetrachloride .ml 5.0 Dry bakers yeast, Type 1821 g 1.5

The head space in one of the fermentation flasks was filled with nitrogen, the other with carbon dioxide, and both flasks were incubated at 37 C. in a'water bath with shaking. Thereafter, the pH of each flask was adjusted to 7.6 with aqueous sodium hydroxide, and to each flask were added 30 ml. of aqueous 2% alpha-ketoglutaric acid solution which had likewise been adjusted to pH 7.6 with aqueous sodium hydroxide. Thefermentation was then resumed under nitrogen and carbon dioxide, as before. The results at the end of ten hours were as follows:

L-Glutamic Acid a-KOtO- glutarate added, pM ./m1.

Atmosphere Time,

hr. Found,

thesize #MJml.

N2 18} 3; CO2 18} i ,8;

Example V The following tests illustrate the eifects of different concentrations of metallic ion enzyme activator. An aqueous fermentation medium having the following composition was prepared.

0.2 M dibasic ammonium phosphate (pH 7.6 with HCl) ml 15.0 Sucrose g... 1.0 Water ml 4.5 Carbon tetrachloride ml. 5.0 Dry bakers yeast, Type 1821 g 3.0

Five portions of the medium were made up, and to the portions were added manganese ion in concentrations ranging from 0 to 0.01 molar, together with about M./ml. of alpha-ketoglutarate. The mixture was incubated in a nitrogen atmosphere on a reciprocal shaker at 37 C. for 17 hours. The results were as follows:

a-Keto- L-Glutamio Aied glutarate Mn ion Concentration,

Molar a 8 pM .lml.

Synthesized,

Conversion, yIVI ./m1.

percent Example VI The following test illustrate the results of employing various alkaline materials for pH adjustment. An aqueous fermentation medium having the following composition was prepared:

0.2 M dibasic ammonium phosphate (pH 7.6, with HCl) ml 30.0 MnSO, (0.003 M) "ml-.. 0.2 Water ml 9.8 Dry bakers yeast, Type 1821 g 6.0 Sucrose g 2.0 Carbon tetrachloride .ml 10.0

The medium was incubated 2 hours at 37 C., and then 60 ml. of aqueous 2% alpha-kctoglutaric acid (adjusted to pH 7.6 with a selected base) were added. The complete mixture was adjusted to pH 7.6 with the selected base and returned to the 37 C. incubator. Samples were removed foranalysis at 0, 24, and 48 hours. The results were as follows:

From the foregoing data it will be observed that potassium hydroxide is an especially advantageous base for use in pH adjustment.

Example VII The ollbw te .i t e h n ta mtnf ma acid according to our invention. Three portions of a fermentation medium were prepared as described in Example I, adjusted to pHv 7.6 with NaOH, and preincubated 2 hours at 37 C. under a nitrogen atmosphere. To the respective media were then added 2% aqueous solutions of oxalacetic acid, fumaric acid, and malic acid neutralized to pH 7. 6 NaOH, and the fermentation was continued, th mples being shaken on a reciprocal shake under nitrogen. I a k Analysis of samples at and 24 hours showed that the aspartic acid level had doubled when oxalac etate was the substrate and had increased time when either fumarate or malate was the substrate. I

Having now thus fully described and illustrated the character of the instant invention, what is desired to be secured by Letters Patent is: h a

1.111 a process for preparing an alpha-amino" dicarboxylate selected from the group consisting of L-a spart'ate and L-gl'utaniate by" fermenting an appropriate alpha-keto dicarboxylate' precursor compound with yeast, the improvement which comprises carrying out said fermentation with whole yeast cells in the presence of a lipid solvent.

2. The process of claim 1 wherein thefermentation medium, prior to the introduction of alpha-keto dicarboxylate, is subjected to a preincubation of about 1 to about 10 hours. I

3. The process of claim 1 wherein said fermentation is carried out in the absence of free oxygen.

4. The process of claim 3 wherein said fermentation is carried out under an atmosphere of carbon dioxide.

5. The process of claim 1 wherein said lipid solvent is carbon tetrachloride.

6. In a process for preparing an alpha-amino dicarboxylate selected from the group consisting of L asp artate and L-glutamate by fermenting an appropriate alpha-keto dicarboxylate precursor compound with yeast and recovering the alpha-amino dicarboxylate from the reaction mixture; the improvement which comprises carrying out said fermentation with whole yeast cells in the presence of a sufiicient quantity of a lipid solvent to dissolve free lipids substantially completely from said waste gas cent.

7. A process as in claim 6 which the fermentation S is carried out at a temperature between about 20 and about50 C. p

8. A process as in claim 6" in which the fermentation is carried out in the prose of ammonium ions, phosphate ions, and a fermentab arhohydrate.

9. A process as in claim 8 in which the fermentation is carried out the presence of an enzyme activator capableof enhancing the activity of the yeast.

10. A process as in claim 9 in whichthe enzyme activator is manganous sulfate and the temperature of the fermentation is maintained, between about and about C.

11. A process for preparing L-aspartic acid which comprises fermenting a compound selected'from the group consisting of oxalacetic acid, fumaricacid, and malic acid in an aqueous fermentatidn medium with whole yeast cells in the presence of a lipid solvent and recovering L-aspartic acid from the fermented reaction product 12. A process for prep fgL-gmamie and which comprises fermenting alpha-temperate acid in" an aqu ous fermentation medium with whole yeast cells in the presence of a lipid solvent and recovering" L-gl'utarnic acid from the reaction product mixture. 7

V 13. A process for preparing L-glu 'tam'ic acid which comprises fermenting alpha-ketoglutaric acid at a temperature between about" 35 and -about 40 C. in an aqueous fermentation medium in the presence of ammonium ions; phosphate ions, carbon tetrachloride, 'sucrbse, and manganous sulfate, and recovering L glutathic acid from the reaction product mixture.

14. A processfor preparing an lplia afriino' dicarboxyl'ate selected'from the grou consisting of L aspartate and L-glutamate which comprises preparing armature: tion medium containing ammonium ions, phosphate ions, a lipid solvent, 21 fermentable carbohydrate, and whole yeast cells, incubating the resulting medium for at least about 15 minutes, adding an appropriate alpha lietodicarboxylate precursor compound to the incubated medium, again incubating the resulting medium for at least about 15 minutes, and recovering said alpha-amino dicarboxyiate from the fermentation reaction product mixture.

15. A process as in claim 14 in which the fermentation medium, prior to the second incubatioh period, is adjusted to a pH between about 6 and about 8 by addition of potassium hydroxide. p

16. A process for preparing L-glutamic acid which comprises preparinga fermentation medium containing ammonium ions, phosphateions a lipid solvent, a fermentable carbohydrate, and Whole yeast cells, incubating the result ing medium at a temperature between about 20 and about C. for a period of about 1 to about 10 hours, adding alpha-ketoglutarate thereto, adjusting the pH of the medium to between about 6 and about 8, again incubating the medium in the substantially complete absence of free oxygen at a temperature between about 20 and about 50 C. for a period of about 15 minutes to about 20 hours, and recovering L-glutamic acid from the resulting fermentation liquor.

References Cited in the file of this patent UNITED STATES PATENTS Smythe et al. June 5:, 19 56 Smythe et al. Dec. 4, 1956 l l l 

